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Results for “Ribosome Profiling”

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At least 235 records · Page 13Linked to original sources

PubMed2005

Pulmonary mycetoma caused by an atypical isolate of Paecilomyces species in an immunocompetent individual: case report and literature review of Paecilomyces lung infections.

The first case of pulmonary fungus ball caused by Paecilomyces species is reported. The diagnosis was established by isolation of the fungus in culture from specimens obtained by percutaneous transthoracic needle aspiration in an immunocompetent individual. The isolate was atypical, as it failed to produce fruiting structures on routine mycological media. Identification was achieved by sequencing polymorphisms of the internal transcriber spacer regions of the ribosomal DNA. The antifungal susceptibility profile was also determined. This report underscores the increasing importance of Paecilomyces species in human infections. Paecilomyces should be included among the etiological agents of pulmonary mycetoma.

Adult↗

PubMed1982

Variations in the activity of nucleolar organizers in different tissues, demonstrated by silver staining of human normal and leukemic cells.

A simple silver-staining technique that demonstrates those nucleolar organizing regions of metaphase chromosomes which are transcriptionally active during the preceding interphase (AgNORs) has been applied to cells obtained from the bone marrow and mitogen-stimulated peripheral blood lymphocyte cultures of hematologically normal individuals and patients with various forms of leukemia. In the majority of bone marrow cells from the normal controls and many of the patients, the number of cells with detectable AgNORs, and the staining intensities in those cells which were Ag+, were markedly reduced compared with the levels found in blood lymphocytes. The numbers of cells having satellite associations and the numbers of chromosomes participating in these associations also generally reflected the proportions of AgNORs present. When patterns of bone marrow silver staining were compared between patients with leukemia, distinct differences were found which could be correlated with cytology. It is suggested that different cell types have characteristic AgNOR staining profiles, reflecting specific regulation of ribosomal RNA synthesis in particular cell lineages. AgNOR staining may indicate, therefore, the predominant cell types that divide in the bone marrows of patients with different forms of leukemia.

Bone Marrow Cells↗

PubMed1986

Wandering cells in the nephron of the mudskipper Periophthalmus koelreuteri (Pallas).

Wandering cells that infiltrate between the principal cells of the nephron of the mudskipper Periophthalmus koelreuteri were examined by transmission electron microscope. These cells were found at various levels between the tubule cells and were more abundant in the proximal and collecting segments. These cells have cytoplasmic processes and large spherical nuclei. Their cytoplasm appears electron dense and contains many lysosomes, rough endoplasmic reticulum, free ribosomes, some mitochondria and Golgi profiles. Their ultrastructural features indicate that they may be phagocytic.

Animals↗

PubMed1976

The use of mercurated nucleoside triphosphate as a probe in transcription studies in vitro.

1. Purified form A RNA polymerase and the endogenous, nuclear form A RNA polymerase are shown to incorporate 5-mercuri-uridine 5'-triphosphate (Hg-UTP) into RNA in vitro. The Km for both Hg-UTP and UTP are in the region of 10 muM for the purified enzyme. 2. The RNA products formed in nucleoli by endogenous RNA polymerase A have similar base compositions (G + C-rich) whether UTP or Hg-UTP is provided as the substrate in vitro. 3. Sulphydryl-Sepharose chromatography of RNA synthesised in vitro by nucleoli allows separation of this material from the endogenous RNA, when the former is synthesised in the presence of Hg-UTP. 4. In-vitro-synthesised nucleolar RNA hybridises with cot profiles similar to 28-S ribosomal RNA, when made with either Hg-UTP or UTP. 5. Hybridisation studies using DNA excess suggest that little competition occurs between the in vitro transcripts and the endogenous nucleolar RNA. 6. Size analysis of in vitro transcripts show that although some degradation occurs during isolation, purification and hybridisation of the RNA species, most of the RNA remains larger than 5 S throughout.

Animals↗

PubMed1975

Inhibition of protein synthesis in intact HeLa cells.

Polysome analysis has proved to be a sensitive probe for the mode of action of inhibitors of protein synthesis in intact HeLa cells. To classify the active compounds as inhibitors of initiation, elongation, or termination, their effects on the cellular polyribosome pattern were compared under three conditions. These conditions tested (i) their direct effect on the polyribosome profile; (ii) their effect on ribosome run-off produced by hypertonicity; and (iii) their effects on recovery from hypertonicity. Using this technique, diacetoxyscirpenol, 2-(4-methyl-2,6-dinitroanilino)-N-methylpropionamide, and three alkaloids, harringtonine, isoharringtonine, and homoharringtonine, were found to be inhibitors of initiation. Polysome analysis indicated that in HeLa cells 7.8 x 10(-7) M pactamycin, which inhibited protein synthesis 94%, interfered with elongation as well as initiation under these conditions. Emetine, anisomycin, cycloheximide, and trichodermin each gave polysome patterns consistent with inhibition of elongation. Fusidic acid and aurintricarboxylic acid inhibited incorporation of [(14)C]leucine into intact HeLa cells, but polysome analysis did not localize any specific inhibitory effects to the initiation, elongation, or termination steps of protein synthesis. The use of specific inhibitors of initiation of protein synthesis has indicated that most, if not all, mammalian messenger ribonucleic acids contain a single initiation site.

Amino Acids↗

PubMed1999

Isolation from the Sorghum bicolor mycorrhizosphere of a bacterium compatible with arbuscular mycorrhiza development and antagonistic towards soilborne fungal pathogens.

A gram-positive bacterium with antagonistic activity towards soilborne fungal pathogens has been isolated from the mycorrhizosphere of Sorghum bicolor inoculated with Glomus mosseae. It has been identified as Paenibacillus sp. strain B2 based on its analytical profile index and on 16S ribosomal DNA analysis. Besides having antagonistic activity, this bacterium stimulates mycorrhization.

Base Sequence↗

PubMed2026

Effects of Fecal Microbiota Transplantation on Intestinal Microbial Characteristics and Clinical Phenotypes in Patients with Parkinson's Disease.

Alterations in the gut microbiota have been associated with Parkinson's disease (PD), but longitudinal microbial changes after fecal microbiota transplantation (FMT) and their clinical associations remain poorly understood. This single-center retrospective observational study included 6 patients with PD, stratified into high- and low-severity subgroups based on disease duration (>6 years vs ≤6 years). Thirty-six fecal samples were collected before FMT and monthly for five months afterward. Microbial diversity, community structure, taxonomic composition, and predicted functional profiles were assessed using 16S ribosomal RNA gene sequencing. Analyses included alpha and beta diversity, taxonomic abundance, linear discriminant analysis effect size, Tax4Fun2-based functional prediction, and Spearman rank correlations between microbial features and clinical indicators. Descriptive analyses indicated differences in microbial richness, diversity, community structure, and predicted functions between severity subgroups and across post-FMT time points. At baseline, the low-severity subgroup had greater microbial richness and diversity than the high-severity subgroup, with relatively higher abundances of taxa including Bifidobacterium and Lactobacillus. One month after FMT, richness and diversity increased from baseline in the high-severity subgroup, accompanied by changes in taxonomic composition. Both subgroups showed time-associated variation in microbial diversity and predicted Kyoto Encyclopedia of Genes and Genomes pathway enrichment after FMT. Predicted functions included carbohydrate and amino acid metabolism, secondary metabolite biosynthesis, membrane transport, and signal transduction. Several operational taxonomic units correlated with indicators of motor impairment, constipation, sleep quality, functional status, and neuropsychiatric symptoms. FMT was therefore associated with longitudinal changes in gut microbial diversity, composition, and predicted functions, and specific microbial features were associated with motor and non-motor indicators. Given the small retrospective cohort, these findings are preliminary and warrant confirmation in larger controlled studies. Future studies should determine whether these microbial alterations are reproducible, persist beyond five months, reflect donor engraftment, and correspond to measurable clinical improvement after transplantation in PD.

Humans↗

PubMed1989

[Ultrastructural and cytochemical studies of malignant histiocytosis].

The internal and surface ultrastructure and cytochemistry (MPO, AP) of the histiocytes from 16 patients were studied. The results showed that poorly differentiated histiocyte was irregular in cell contour with various projections. All had more or less neoplastic histiocytes in which numerous vacuoles and blisters along peripheral plasma membrane were seen. The neoplastic cells had numerous lysosomes, abundant short strands of rough endoplasmic reticulum, and free and poly-ribosomes in the cytoplasma. Long profiles and ring of RER as well as expansive forms were seen in 4 cases. The mitochondria were generally normal although an occasional elongated form and their cristae showed destroyed architecture in 6 cases. Occasionally, perinuclear bundles of microfilaments and prominent Golgi apparatus were seen. The nuclei of the neoplastic histiocytes were irregularly shaped and most had one or two huge nucleoli. Circular nuclear bodies were often seen. The ultrastructural cytochemistry of the neoplastic histiocytes showed: MPO was weakly positive in about 1/4 to 1/2 of the cells. AP reaction in all of the neoplastic histiocytes was always strongly positive located in the nuclear membrane. RER, lysosomes granules and Golgi apparatus. Under SEM, various surface characteristics of the neoplastic histiocytes were seen such as bleb-covered surface; bleb-ruffled surface, and ruffled surface.

Adult↗

PubMed1985

The nuclear binding of estradiol stimulates ribonucleoprotein transport in the rat uterus.

An inverse relationship exists in the rat uterus between the endogenous estradiol concentration and the rate of accumulation within the nuclei of ribonucleoproteins (RNP) that bind estradiol. Exposure of the uterine nuclei to physiological concentrations of estradiol, either in vivo or in vitro, results in the hormone binding to the nuclear RNP and the transport of the RNP-estradiol complex from the nuclei. Evidences suggest that the RNP-estradiol complex, thus released upon in vivo exposure of the nuclei to estradiol, associates with the ribosomes forming polysomes. The polysome profiles show that the hormone-binding activity is mainly associated with the rapidly sedimenting polysomal fractions.

Adenosine Triphosphate↗

PubMed1998

Fingerprinting of Flavobacterium psychrophilum isolates by ribotyping and plasmid profiling.

Flavobacterium psychrophilum is the agent of cold-water disease and rainbow trout fry syndrome in salmonid fish worldwide. Ribosomal RNA gene restriction patterns (ribotypes) and plasmid profiles were determined on a collection of 85 strains isolated from different countries and fish species. Several ribotypes were obtained by using the restriction endonucleases Hinc II and Pvu II. Computer analysis of the ribotypes revealed that some of them were clearly associated with the fish species from which the strains were isolated, whereas no correlation with the geographical origin was found. Most of the strains harboured at least one plasmid and several different plasmid profiles were observed, even among strains sharing the same ribotype. These methods, used alone or in combination with other typing techniques, can be considered powerful tools for the epidemiological tracing of F. psychrophilum infections.

Animals↗

PubMed1980

Fractionation of native and reconstituted chromatin by digesting with deoxyribonuclease ii.

Native and reconstituted chromatin from Ehrlich ascites tumor cells were fractionated into template-active and inactive fractions by the DNAase II/Mg2+-solubility method of Gottesfeld et al. (Gottesfeld, J.M., Garrard, W.T., Bagi, G., Wilson, R.F. and Bonner, J. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 2193-2197). The Mg2+-soluble (template-active) fractions were compared in respect to sedimentation behavior in sucrose gradients and the relative content of specific transcribed (ribosomal) and non-transcribed (satellite) DNA sequences. It was found that the Mg2+-soluble fraction of the native chromatin was enriched in ribosomal DNA while almost completely devoid of satellite DNA; the nucleoprotein monomer of this fraction sedimented in sucrose gradient at 14 S. Similar-results were obtained if chromatin was fractionated in the presence of 3 M urea. With reconstituted chromatin, however, neither the sedimentation profile, nor the relative content of ribosomal and satellite DNA sequences were recovered, thus indicating that reconstitution did not yield nucleoprotein structurally similar to native chromatin.

Animals↗

PubMed2002

One-step affinity purification of the yeast ribosome and its associated proteins and mRNAs.

We describe a one-step affinity method for purifying ribosomes from the budding yeast Saccharomyces cerevisiae. Extracts from yeast strains expressing only C-terminally tagged Rpl25 protein or overexpressing this protein in the presence of endogenous Rpl25p were used as the starling materials. The purification was specific for tagged 60S subunits, and resulted in the copurification of 80S subunits and polysomes, as well as ribosome-associated proteins and mRNAs. Two of these associated proteins, Mpt4p and Asc1p, were nearly stoichiometrically bound to the ribosome. In addition, the degree of mRNA association with the purified ribosomes was found to reflect the mRNA's translational status within the cell. The one-step purification of ribosome and its associated components from a crude extract should provide an important tool for future structural and biochemical studies of the ribosome, as well as for expression profiling of translated mRNAs.

Base Sequence↗

PubMed2007

Development of thermodynamic models for simulating probe dissociation profiles in fluorescence in situ hybridization.

Stringency in ribosomal RNA (rRNA)-targeted fluorescence in situ hybridization (FISH) is typically adjusted with formamide, and the optimum formamide concentration at which the probe can hybridize with the target rRNA, but not with rRNAs with mismatches, is to be found experimentally. This is a difficult task when target or closest non-target organisms are not available in pure culture, or when there are numerous non-targets of concern. The objective of this work was to formulate mechanistic models capable of simulating the effect of formamide on probe dissociation. Using a previously described equilibrium model of FISH [Yilmaz and Noguera (2004) Applied and Environmental Microbiology 70(12):7126-7139] as the basis, the effect of formamide on free energy changes of probe-target duplex formation (DeltaG(1)(0)) and folding of target region (DeltaG(3)(0)) was simulated to be linear, and models differing in the definitions of the slopes of these relationships (m(1) and m(3)) were calibrated using experimental dissociation profiles for 27 probes targeting the 16S rRNA of Escherichia coli (E. coli). A good level of predictive power was obtained when m(1) was linearly related to probe length and when m(3) was made proportional to DeltaG(3)(0). The effect of single mismatches on probe dissociation with formamide was also studied, although at a preliminary level. The expected changes in DeltaG(1)(0) with the introduction of mismatches were not sufficient to capture the overall trends of mismatched dissociation profiles. In conclusion, this study offers the first theoretical method to calculate dissociation profiles for perfectly matched probes, and suggests a direction to systematically evaluate the effect of formamide on mismatched probes.

Base Pair Mismatch↗

PubMed2004

New strategy for the representation and the integration of biomolecular knowledge at a cellular scale.

The combination of sequencing and post-sequencing experimental approaches produces huge collections of data that are highly heterogeneous both in structure and in semantics. We propose a new strategy for the integration of such data. This strategy uses structured sets of sequences as a unified representation of biological information and defines a probabilistic measure of similarity between the sets. Sets can be composed of sequences that are known to have a biological relationship (e.g. proteins involved in a complex or a pathway) or that share similar values for a particular attribute (e.g. expression profile). We have developed a software, BlastSets, which implements this strategy. It exploits a database where the sets derived from diverse biological information can be deposited using a standard XML format. For a given query set, BlastSets returns target sets found in the database whose similarity to the query is statistically significant. The tool allowed us to automatically identify verified relationships between correlated expression profiles and biological pathways using publicly available data for Saccharomyces cerevisiae. It was also used to retrieve the members of a complex (ribosome) based on the mining of expression profiles. These first results validate the relevance of the strategy and demonstrate the promising potential of BlastSets.

Computational Biology↗

PubMed2001

Depletion and deletion analyses of eucaryotic translation initiation factor 1A in Saccharomyces cerevisiae.

Translation initiation factor eIF1A is a highly conserved, small, acidic protein that is required for cell growth in yeast. Biochemical studies in vitro implicate eIF1A in dissociating ribosomes, promoting methionyl-tRNA(i) binding to 40S ribosomal subunits, scanning of mRNAs and recognizing the AUG initiation codon. To elucidate the pleiotropic functions of eIF1A in vivo, the factor was depleted by placing its gene behind the repressible GAL1 promoter. After Saccharomyces cerevisiae cells were shifted to glucose medium, depletion of eIF1A was seen after 3-4 generations, corresponding with cessation of cell growth. Polysome profiles of the depleted strain showed ribosome run-off from mRNAs, indicating that eIF1A is involved in the initiation phase of translation. A decrease in free 40S ribosomes and an apparent increase in free 60S ribosomes were attributed to the formation of 40S subunit dimers. The result suggests that one of the functions of eIF1A is to prevent formation of 40S dimers. Mutant forms of eIF1A lacking either the positively charged N-terminal region or the negatively charged C-terminal region were constructed and tested for their ability to confer cell growth as the sole source of eIF1A. Either deletion supports cell growth, albeit at a slower rate, and causes a reduction in polysomes, although eIF1A lacking the N-terminal region is more deleterious. Therefore the charged terminal regions contribute to, but are not absolutely essential for, eIF1A function.

Amino Acid Sequence↗

PubMed2001

pH-dependent conformational flexibility within the ribosomal peptidyl transferase center.

A universally conserved adenosine, A2451, within the ribosomal peptidyl transferase center has been proposed to act as a general acid-base catalyst during peptide bond formation. Evidence in support of this proposal came from pH-dependent dimethylsulfate (DMS) modification within Escherichia coli ribosomes. A2451 displayed reactivity consistent with an apparent acidity constant (pKa) near neutrality, though pH-dependent structural flexibility could not be rigorously excluded as an explanation for the enhanced reactivity at high pH. Here we present three independent lines of evidence in support of the alternative interpretation. First, A2451 in ribosomes from the archaebacteria Haloarcula marismortui displays an inverted pH profile that is inconsistent with proton-mediated base protection. Second, in ribosomes from the yeast Saccharomyces cerevisiae, C2452 rather than A2451 is modified in a pH-dependent manner. Third, within E. coli ribosomes, the position of A2451 modification (N1 or N3 imino group) was analyzed by testing for a Dimroth rearrangement of the N1-methylated base. The data are more consistent with DMS modification of the A2451 N1, a functional group that, according to the 50S ribosomal crystal structure, is solvent inaccessible without structural rearrangement. It therefore appears that pH-dependent DMS modification of A2451 does not provide evidence either for or against a general acid-base mechanism of protein synthesis. Instead the data suggest that there is pH-dependent conformational flexibility within the peptidyl transferase center, the exact nature and physiological relevance of which is not known.

Base Sequence↗

PubMed2005

Role of mammalian target of rapamycin signaling in compensatory renal hypertrophy.

Loss of functioning nephrons stimulates the growth of residual kidney tissue to augment work capacity and maintain normal renal function. This growth largely occurs by hypertrophy rather than from hyperplasia of the remaining nephrons. The signaling mechanisms that increase RNA and protein synthesis during compensatory renal hypertrophy are unknown. This study found that the remaining kidney hypertrophied 42% by 16 d after unilateral nephrectomy (UNX) in DBA/2 mice. Immunoblotting analysis revealed increased phosphorylation of the 40S ribosomal protein S6 (rpS6) and the eukaryotic translation initiation factor (eIF) 4E-binding protein 1 (4E-BP1), the two downstream effectors of the mammalian target of rapamycin (mTOR). The highly specific mTOR inhibitor rapamycin blocked UNX-increased phosphorylation of both rpS6 and 4E-BP1. UNX increased the content of not only 40S and 60S ribosomal subunits but also 80S monosomes and polysomes in the remaining kidney. Administration of rapamycin decreased UNX-induced polysome formation and shifted the polysome profile in the direction of monosomes and ribosomal subunits. Pretreatment of the mice with rapamycin inhibited UNX-induced hypertrophy. These studies demonstrate that activation of the mTOR signaling pathway in the remaining kidney after UNX plays an essential role in modulating RNA and protein synthesis during development of compensatory renal hypertrophy.

Adaptation, Physiological↗

PubMed1977

Mitochondrial and cytoplasmic ribosomes from mammalian tissues. Further characterization of ribosomal subunits and validity of buoyant-density methods for determination of the chemical composition and partial specific volume of ribonucleoprotein particles.

1. At 0-4 degrees C mitochondrial ribosomes (55S) dissociate into 39S and 29S subunits after exposure to 300mm-K(+) in the presence of 3.0mm-Mg(2+). When these subunits are placed in a medium containing a lower concentration of K(+) ions (25mm), approx. 75% of the subparticles recombine giving 55S monomers. 2. After negative staining the large subunits (20.3nm width) usually show a roundish profile, whereas the small subunits (12nm width) show an elongated, often bipartite, profile. The dimensions of the 55S ribosomes are 25.5nmx20.0nmx21.0nm, indicating a volume ratio of mitochondrial to cytosol ribosomes of 1:1.5. 3. The 39S and 29S subunits obtained in high-salt media at 0-4 degrees C have a buoyant density of 1.45g/cm(3); from the rRNA content calculated from buoyant density and from the rRNA molecular weights it is confirmed that the two subparticles have weights of 2.0x10(6) daltons and 1.20x10(6) daltons; the weights of the two subunits of cytosol ribosomes are 2.67x10(6) and 1.30x10(6) daltons. 4. The validity of the isodensity-equilibrium-centrifugation methods used to calculate the chemical composition of ribosomes was reinvestigated; it is confirmed that (a) reaction of ribosomal subunits with 6.0% (v/v) formaldehyde at 0 degrees C is sufficient to fix the particles, so that they remain essentially stable after exposure to dodecyl sulphate or centrifugation in CsCl, and (b) the partial specific volume of ribosomal subunits is a simple additive function of the partial specific volumes of RNA and protein. The RNA content is linearly related to buoyant density by the equation RNA (% by wt.)=349.5-(471.2x1/rho(CsCl)), where 1/rho(CsCl)=[unk](RNP) (partial specific volume of ribonucleoprotein). 5. The nucleotide compositions of the two subunit rRNA species of mitochondrial ribosomes from rodents (42% and 43% G+C) are distinctly different from those of cytoplasmic ribosomes.

Animals↗